PROJECT SUMMARY Mycobacterium tuberculosis (Mtb) is the causative pathogen in tuberculosis (TB). TB is the leading cause of death from infectious disease globally and is especially prevalent in individuals infected with HIV. While normally thought of as a respiratory disease, TB also infects other organ systems. Infection of the central nervous system (CNS-TB) is the most severe form of the disease, and has a mortality rate of nearly 50%, despite aggressive clinical intervention. CNS-TB is associated with severe neurological dysfunction including cranial nerve palsies, cognitive impairment, stroke, and seizures. The brain?s molecular, cellular, and network level response to CNS-TB is almost completely unknown. We hypothesize CNS-TB leads to significant activation of neuroinflammatory signaling, as well as glial and neuronal dysfunction. The risk of developing CNS-TB is markedly increased under conditions of immune suppression, evident in HIV infected individuals, who have a higher occurrence of TB meningitis (TBM). A regulated tumor necrosis factor (TNF) response is a critical feature of immune competence necessary for protection against TB, and is lost in progressive HIV infection. The importance of a proper TNF response is clinically validated by the reactivation of TB in patients on anti-TNF treatment for autoimmune diseases. We recently reported that TNF deficiency (a model of immune suppression) in mice (TNF-/-), promotes CNS-TB infection, a hyper-inflammatory response, gross brain pathology, and mortality. We will utilize a realistic mouse model of CNS-TB in which active Mycobacterium tuberculosis (Mtb) will be injected into the brains of control and immune compromised (TNF-/-) mice to study how resident CNS cell respond. Although Mtb primarily infects microglia and astrocytes, human and mouse neurons also act as host cells for Mtb. Therefore, multiple CNS cells react both directly and indirectly to brain infection, creating a complex cellular- and tissue-level response. To deal with this complexity, we will utilize single cell RNA-seq, a cutting edge genomic approach, which allows transcriptional analysis of the response to CNS-TB on a cell-by-cell basis. This approach enables the identification of individual types of cells, for example astrocytes, and analysis of their unique transcriptional response. In addition, single cell RNA-seq allows investigation of the heterogeneity of the cellular response to CNS-TB by analyzing individual cells. We will utilize single cell RNA-seq to determine how the lack of a proper immune response regulates neuroinflammatory signaling and leads to broad-scale disruption of CNS resident cells in a mouse model of CNS-TB. In doing so, we will establish a partnership between The University of Cape Town and Tufts University. Emphasis will be placed on training South African scientists in neuro- immune interactions, single cell RNA-seq, and advanced genomic analysis approaches, thereby helping develop robust research infrastructure in South Africa.